2J6U

Ternary complex of Sulfolobus solfataricus Dpo4 DNA polymerase, O6- methylguanine modified DNA, and dGTP.


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.265 
  • R-Value Work: 0.217 
  • R-Value Observed: 0.217 

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Ligand Structure Quality Assessment 


This is version 1.2 of the entry. See complete history


Literature

Sulfolobus Solfataricus DNA Polymerase Dpo4 is Partially Inhibited by "Wobble" Pairing between O6- Methylguanine and Cytosine, But Accurate Bypass is Preferred.

Eoff, R.L.Irimia, A.Egli, M.Guengerich, F.P.

(2007) J Biol Chem 282: 1456

  • DOI: https://doi.org/10.1074/jbc.M609661200
  • Primary Citation of Related Structures:  
    2J6S, 2J6T, 2J6U

  • PubMed Abstract: 

    We examined the effect of a single O6-methylguanine (O6-MeG) template residue on catalysis by a model Y family polymerase, Dpo4 from Sulfolobus solfataricus. Mass spectral analysis of Dpo4-catalyzed extension products revealed that the enzyme accurately bypasses O6-MeG, with C being the major product (approximately 70%) and T or A being the minor species (approximately 20% or approximately 10%, respectively), consistent with steady-state kinetic parameters. Transient-state kinetic experiments revealed that kpol, the maximum forward rate constant describing polymerization, for dCTP incorporation opposite O6-MeG was approximately 6-fold slower than observed for unmodified G, and no measurable product was observed for dTTP incorporation in the pre-steady state. The lack of any structural information regarding how O6-MeG paired in a polymerase active site led us to perform x-ray crystallographic studies, which show that "wobble" pairing occurs between C and O6-MeG. A structure containing T opposite O6-MeG was solved, but much of the ribose and pyrimidine base density was disordered, in accordance with a much higher Km,dTTP that drives the difference in efficiency between C and T incorporation. The more stabilized C:O6-MeG pairing reinforces the importance of hydrogen bonding with respect to nucleotide selection within a geometrically tolerant polymerase active site.


  • Organizational Affiliation

    Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.


Macromolecules

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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
DNA POLYMERASE IV358Saccharolobus solfataricus P2Mutation(s): 0 
EC: 2.7.7.7
UniProt
Find proteins for Q97W02 (Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2))
Explore Q97W02 
Go to UniProtKB:  Q97W02
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ97W02
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
5'-D(*GP*GP*GP*GP*GP*AP*AP*GP*GP*AP *TP*TP*CP*T)-3'B [auth P]14synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 3
MoleculeChains LengthOrganismImage
5'-D(*TP*CP*AP*C G32P*GP*AP*AP*TP*CP*CP *TP*TP*CP*CP*CP*CP*C)-3'C [auth T]18synthetic construct
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.265 
  • R-Value Work: 0.217 
  • R-Value Observed: 0.217 
  • Space Group: P 21 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 92.318α = 90
b = 102.285β = 90
c = 52.485γ = 90
Software Package:
Software NamePurpose
CNSrefinement
HKL-2000data reduction
HKL-2000data scaling
CNSphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2006-11-22
    Type: Initial release
  • Version 1.1: 2015-10-21
    Changes: Derived calculations, Non-polymer description, Other, Refinement description, Source and taxonomy, Structure summary, Version format compliance
  • Version 1.2: 2023-12-13
    Changes: Data collection, Database references, Derived calculations, Other, Refinement description